This grant proposes to generate protein-based, genetically-encodable sensors for the primary excitatory neurotransmitter glutamate and to express these fusion proteins in situ to study spatiotemporal aspects of excitatory synaptic signaling. A series of constructs based on fusion of a neuronal glutamate transporter (EAAT3 or EAAT4) and ion-sensitive variants of GFP will be generated. Sensor constructs will be evaluated in three stages. First, each will be characterized in HEK cells to establish whether glutamate elicits optical signals, and if so to determine the pH and chloride sensitivity and the kinetic properties of those signals (Aim 1). Next, viable constructs will be inserted into viral vectors suitable for expression in rat brain. The ability of constructs to generate optical signals in response to excitatory synaptic activity will be tested (Aim 2). Combining optical and electrophysiological measurements, constructs will be used to map the spatial pattern of active synaptic inputs to neurons under various conditions (Aim 3). Together these experiments will develop and establish the practical usefulness of a class of genetically-encodable indicators of excitatory synaptic transmission. Once developed, such indicators are expected to have an important impact on the study of information processing within neural circuits, allowing minimally invasive measurement of subthreshold synaptic signals. [unreadable] [unreadable] [unreadable]